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Abstract

Background: Vaccinia virus gene B1R encodes a serine/threonine protein kinase. In vitro this protein kinase phosphorylates ribosomal proteins Sa and S2 and vaccinia virus protein H5R, proteins that become phosphorylated during infection. Nothing is known about the sites phosphorylated on these proteins or the general substrate specificity of the kinase. The work described is the first to address these questions. Results:Vaccinia virus protein H5R was phosphorylated by the B1R protein kinase in vitro, digested with V8 protease, and phosphopeptides separated by HPLC. The N-terminal sequence of one radioactively labelled phosphopeptide was determined and found to correspond to residues 81-87 of the protein, with Thr-84 and Thr-85 being phosphorylated. A synthetic peptide based on this region of the protein was shown to be a substrate for the B1R protein kinase, and the extent of phosphorylation was substantially decreased if either Thr residue was replaced by an Ala. Conclusions:We have identified the first phosphorylation site for the vaccinia virus B1R protein kinase. This gives important information about the substrate-specificity of the enzyme, which differs from that of other known protein kinases. It remains to be seen whether the same site is phosphorylated in vivo.

Orthopoxvirus Targets for the Development of Antiviral Therapies

“…The H5R gene encodes a 203 aa phosphoprotein that has been shown to be a transcription factor for late genes [88]. Its function in the replication complex is currently unclear, however B1R is known to associate with and phosphorylate this protein [89]. D5R encodes a 785 aa protein with a migration rate of 90 kDa.…”

“…The H5R product in VV is a natural substrate of this enzyme [138] and amino acids Thr-84 and Thr-85 were shown to be specifically phosphorylated [89]. Both H5R and B1R and are present in virosomes [138] and co-localize to punctate sites in the cytoplasm that are precursors to sites of viral DNA synthesis [139].…”